Acute pancreatitis (AP) is initiated by injury to pancreatic acinar cells resulting in zymogen activation. This initial insult induces a rapid sterile inflammatory response (SIR) characterized by edema, cellular infiltrate, and further acinar cell death. Many lines of evidence point towards the SIR having a vital role in pancreatic damage, but the crucial link between acinar cell injury and induction of the SIR has not been identified. Our preliminary data demonstrate that in caerulein-induced acute pancreatitis: i. In the absence of TLR9 or the inflammasome components ASC, NLRP3, and caspase-1 there is reduced SIR and acinar cell injury. ii. Inhibitors of TLR9 can reduce the SIR and acinar cell death. iii. TLR9 expression is localized to resident immune, endothelial, and pancreatic duct cells in vivo. We hypothesize that initial acinar cell injury releases damage associated molecular patterns (DAMPs) which are sensed by resident pancreatic cells, resulting in the SIR which increases acinar cell death. Aim 1: Identify the pancreatic DAMP-sensing cell. (a) Bone marrow chimeras will be constructed to identify the relative contribution of immune and parenchymal cells to the SIR. (b) Selective depletion will determine the contributions of individual cell populations to the SIR. Aim 2: Identify which components of inflammasome activation induce acinar cell cytotoxicity. Acinar cells will be isolated and assessed for cell death and zymogen activation in response to (a) IL-1beta and IL-18, and (b) co-culture with the DAMP-sensing cell identified in Aim 1. Aim 3: Identify the regulation of the inflammasome and TLR9 signaling by TLR4 activation in acute pancreatitis. The ability of TLR4 activation to regulate both (a) NLRP3 inflammasome and (b) TLR9 signaling will be assessed in the DAMP-sensing cells identified in Aim 1. The proposed work will identify a detailed cellular and molecular mechanism for the initiation of the inflammatory response in acute pancreatitis. PUBLIC HEALTH RELEVANCE: Acute pancreatitis is a common disease of the pancreas which can rapidly progress into a severe life- threatening inflammatory response and for which there are no effective therapies. How non-infectious insults, such as gallstones, alcohol consumption, drugs and manipulation of the pancreatic duct, which are common triggers of acute pancreatitis, initiate such severe inflammation is poorly understood. This proposal seeks to understand the initiators of the inflammatory response in acute pancreatitis so that therapeutics can be designed to prevent or treat this injurious process.